Method for enzymatic splitting of rutinosides

ABSTRACT

Disclosed is a method for enzymatic splitting of rutinosides, whereby rhamnose and/or corresponding glucopyranosides is/are obtained The inventive method is carried out in the presence of a solvent mixture made up of water and one or several organic solvents.

The invention relates to a process for the enzymatic cleavage ofrutinosides to obtain rhamnose and/or the correspondingglucopyranosides, the reaction being carried out in the presence of asolvent mixture of water and one or more organic solvents.

In the context of the present invention, rutinosides are designated asthose compounds which contain a sugar-free constituent, to which aradical of the

is bonded via a glycosidic bond. For example, the rutinosides areflavonoids having the bisglycosidic unit shown in formula I. Rhamnoseand/or the corresponding glucopyranosides are produced from therutinosides by the process according to the invention. Theglucopyranosides are derived from the rutinosides in that, instead ofthe radical of the formula (I), they contain a radical of the formula(I*)

bonded to the sugar-free constituent. For example, both rhamnose andisoquercetin can be obtained from rutin by the process according to theinvention.

Rhamnose is a monosaccharide which is of widespread occurrence innature, but usually only in small amounts. An important source ofrhamnose is, for example, the glycosidic radicals of natural flavonoidssuch as rutin, from which the rhamnose can be obtained by glycosidecleavage. Rhamnose, for example, plays an important role as a startingsubstance for the preparation of synthetic aromatic substances such asfuraneol.

Isoquercetin is a monoglycosidated flavonoid of the following structuralformula (II)

Flavonoids. (lat. flavus=yellow), which are widespread colorants inplants, are designated as being, for example, glycosides of flavones, towhich the parent structure of flavone (2-phenyl-4H-1-benzopyran-4-one)is common.

The sugar-free constituent of the flavonoids is the so-called aglycone.Isoquercetin is, for example, a glycoside of the aglycone quercetin(2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one), whichdiffers from flavone by the presence of five hydroxyl groups. Inisoquercetin, the carbohydrate radical glucose is bonded to the hydroxylgroup in position 3 of the quercetin. Isoquercetin is designated, forexample, as quercetin 3-O-β-D-glucopyranoside or2-(3,4-dihydroxyphenyl)-3-(β-D-glucopyranosyloxy)-5,7-dihydroxy-4H-l-benzopyran-4-one.However, it is also known, for example, under the name hirsutrin.

Flavonoids and flavonoid mixtures are used, for example, in thefoodstuffs and cosmetics industries and are increasingly gainingimportance there. Particularly monoglycosidated flavonoids such asisoquercetin are distinguished by a good absorption capacity in thehuman body.

An example of a naturally occurring flavonoid having a bisglycosidicunit is rutin, which has the following structural formula (III):

Rutin, like isoquercetin, is likewise a glycoside of the aglyconequercetin where the carbohydrate radical rutinose is bonded to thehydroxyl group in position 3 of the quercetin. The carbohydrate radicalin rutin consists of a glucose unit linked in the 1 and 6 positions anda terminally bonded rhamnose or 6-deoxymannose unit. Rutin isdesignated, for example, as quercetin 3-O-β-D-rutinoside or2-(3,4-dihydroxy-phenyl)3-{[6-O-(6-deoxy-α-mannopyranosyl)-β-D-gluco-pyranosyl]oxy}-5,7-dihydroxy-4H-1-benzopyran-4-one.However, it is also known, for-example, under the names sophorin,birutan, rutabion, tarutin, phytomelin, melin or rutoside.

Rutin, with three molecules of water of crystallization, forms paleyellow to greenish needles. Anhydrous rutin has the properties of a weakacid, becomes brown at 1250° C. and decomposes at 214-2150° C. Rutin,which occurs in many plant species—frequently as an associate of vitaminC —, e.g. in citrus species, in yellow pansies, forsythia and acaciaspecies, various Solanum and Nicotiana species, capers, lime blossom,St. John's wort, tee etc. was isolated from the common rue (Rutagraveolens) in 1842. Rutin can also be obtained from the leaves ofbuckwheat and of the east-asiatic pagoda tree Wei-Fa (Sophora japonica,Farbaceae), which contains 13-27% of rutin.

For the abovementioned reasons, it is desirable to prepare both rhamnoseand monoglycosidated flavonoids from natural raw materials, for examplefrom flavonoids having a bisglycosidic unit. In this connection, forexample, the cleavage of rutinosides to rhamnose and the correspondingglucopyranosides is of interest.

Enzymatically catalyzed preparations of rhamnose are described in theliterature. For example, EP 0 317 033 describes a process for thepreparation of L-rhamnose, with the rhamnosidic bonding of glycosideswhich contain rhamnose bonded in the terminal position being achieved byenzymatic hydrolysis. However, cleavages of this type carried out inaqueous media of glycosides having a bisglycosidic structure of thecarbohydrate radical usually proceed with low selectivity. For example,on account of the bisglycosidic structure of the carbohydrate radical inrutin, a mixture of the two monosaccharides glucose and rhamnose usuallyresults. Moreover, high proportions of the aglycone quercetin and otherundesired by-products usually occur.

In addition, enzymatically catalyzed cleavages of rutin are alsodescribed, for example, in JP 01213293. However, reactions of this typecarried out in aqueous media usually likewise proceed with lowselectivity.

The object was therefore to develop a process for the enzymatic cleavageof rutinosides to obtain rhamnose and/or the correspondingglucopyranosides which avoids or at least diminishes the disadvantagesof the known processes and in particular makes possible a preparation ofrhamnose and the glucopyranosides which is as selective as possible, sothat these products can be prepared in high yield.

Surprisingly, it has now been found that this object is achieved if theprocess for the enzymatic cleavage of rutinosides to obtain rhamnoseand/or the corresponding glucopyranosides is carried out such that thereaction takes place in the presence of a solvent mixture of water andone or more organic solvents.

The process according to the invention is distinguished in particular inthat the cleavage of rutinosides to rhamnose and the correspondingglucopyranosides takes place with high selectivity. Rhamnose and theglucopyranosides are preferably obtained by suitable work-up after theprocess according to the invention. Furthermore, however, either onlyrhamnose or only the glucopyranosides can also be obtained by suitablework-up after the process according to the invention.

The present invention makes available an advantageous process for theenzymatic cleavage of rutinosides to obtain rhamnose and/or thecorresponding gluco-pyranosides. According to this process, therutinoside is contacted with a catalytic amount of an enzyme in asolvent mixture of water and one or more organic solvents. Preferably,the reaction is carried out with thorough mixing, e.g. by stirring.

The reaction is preferably carried out under a nitrogen atmosphere.

Suitable rutinosides for the process according to the invention are, forexample, rutinosides which, as a sugar-free constituent or aglycone,contain a 2-phenyl-4H-1-benzopyran-4-one parent structure which carriesa radical of the formula (I) in position 3 and whose phenyl groups,apart from position 3, can also be mono- or polysubstituted by —OH or—O—(CH₂)_(n)—H, where n is 1 to 8.

n is preferably 1.

The substitution of the 2-phenyl-4H-1-benzopyran-4-one parent structureby —OH and/or —O—(CH₂)_(n)—H preferably occurs in positions 5, 7, 3′and/or 4′.

Particularly preferred rutinosides correspond to formula (IV)

in which R is H (kaempferol rutinoside), OH (rutin) or OCH₃(isorhamnetin rutinoside). Rhamnose and kaempferol glucoside can beobtained from kaempferol rutinoside by the process according to theinvention, rhamnose and isoquercetin from rutin, and rhamnose andisorhamnetin glucoside from isorhamnetin rutinoside. The rutinosiderutin is particularly preferably used.

The invention also relates to the use of kaempferol glucoside,isoquercetin and/or isorhamnetin glucoside in the foodstuffs andcosmetics industries.

The process according to the invention does not need any highly purestarting materials. For example, mixtures of rutinosides can also beused for the process according to the invention. The reaction also takesplace, for example, if the starting material is contaminated with otherflavonoids. It can also be carried out, for example, with mother liquorresidues from rutin production.

Suitable enzymes for the process according to the invention arehydrolases. Hydrolases which have been obtained from the strainPenicillium decumbens are preferably used, in particular the enzymesnaringinase and hesperidinase. The enzyme naringinase is veryexceptionally preferred.

The starting materials and enzymes for the process according to theinvention are commercially obtainable or can be obtained or prepared bymethods which are well known to the person skilled in the art.

Suitable reaction temperatures for the process according to theinvention are temperatures between 15 and 80° C. The process accordingto the invention is preferably carried out at reaction temperatures from30 to 50° C., in particular at reaction temperatures from 35 to 45° C.

If the reaction temperature is too low, the reaction proceeds with aninappropriately slow reaction rate. In contrast, if the reactiontemperature is too high, the enzyme, which is a protein, is denaturedand thus deactivated.

Suitable pHs for the process according to the invention are pHs ofbetween 3 and 8. The process according to the invention is preferablycarried out at pHs from 4.5 to 7, in particular at pHs from 4.8 to 6.8.Furthermore, preferred pHs can however, vary, within the given limitsdepending on the enzyme used. For example, pHs from 6.4 to 6.8 are veryexceptionally preferred when using the enzyme naringinase.

The process is preferably carried out in such a way that the pH isadjusted with the aid of a buffer system. In principle, all customarybuffer systems which are suitable for the adjustment of theabovementioned pHs can be used. Preferably, however, aqueous citratebuffer is used.

Preferably, the preferred temperature and pH ranges are combined, i.e.the reaction is preferably carried out at a reaction temperature of 15to 80° C. and at a pH from 3 to 8, particularly preferably at a reactiontemperature of 30 to 50° C. and at a pH of 4.5 to 7 and particularlypreferably at a reaction temperature of 35 to 45° C. and at a pH of 4.8to 6.8.

The organic solvent(s) present in addition to water include(s) bothorganic solvents which are miscible with water and organic solventswhich are not miscible with water.

Suitable organic solvents for the process according to the invention arenitrites such as acetonitrile, amides such as dimethylformamide, esterssuch as acetic acid esters, in particular methyl acetate or ethylacetate, alcohols such as methanol or ethanol, ethers such astetrahydrofuran or methyl tert-butyl ether and hydrocarbons such astoluene. Preferably, the process according to the invention is carriedout in the presence of one or more of the organic solvents acetic acidesters, methanol, ethanol, methyl tert-butyl ether, toluene.Particularly preferably, the process according to the invention iscarried out in the presence of one or more acetic acid esters, inparticular in the presence of methyl acetate.

Suitable water:organic solvent volume ratios for the process accordingto the invention are ratios of 1:99 to 99:1. Preferably, the processaccording to the invention is carried out with water:organic solventvolume ratios of 20:80 to 80:20, in particular with volume ratios of50:50 to 70:30.

Suitable weight ratios of rutinoside:(water+organic solvent) for theprocess according to the invention are ratios of 0.001:99.999 to 40:60.Preferably, the process according to the invention is carried out withweight ratios of rutinoside:(water+organic solvent) of 0.005:99.995 to20:80, in particular with weight ratios of 0.5:99.5 to 10:90.

Suitable weight ratios of enzyme:rutinoside for the process according tothe invention are ratios of 0.005:99.995 to 50:50. Preferably, theprocess according to the invention is carried out with weight ratios ofenzyme rutinoside of 0.5:99.5 to 30:70, in particular with weight ratiosof 2:98 to 20:80.

The progress or the end of the reaction can be checked, for example, bymeans of thin-layer chromatography (TLC).

After reaction is complete, the reaction mixture consists mainly ofwater, organic solvent, buffer (e.g. sodium citrate), enzyme, smallamounts of unreacted rutinoside, rhamnose, glucopyranoside, smallamounts of the aglycone of rutinoside and, if appropriate, small amountsof glucose. The desired reaction products rhamnose and glucopyranosideare isolated according to customary methods. “Customary work-up” in thecontext of the present invention is understood as meaning the following:

Preferably, the organic solvent is distilled off under reduced pressure.The glucopyranoside hereby crystallizing out, which can contain, forexample, small amounts of the rutinoside and its aglycone, is separatedoff from the remaining reaction mixture, for example by suctionfiltration or filtration under reduced pressure or by centrifuging offthe precipitated crystals. The solid is subsequently washed, preferablywith water, and then dried. The purity of the glucopyranoside obtainedwhen using pure rutinoside is customarily greater than 94%. For furtherpurification, it can be recrystallized, for example, from suitablesolvents, e.g. from water or from solvent mixtures consisting of tolueneand methanol or consisting of water and methyl acetate.

Water, buffer, enzyme, small amounts of rutinoside, small amounts of itsaglycone and, if appropriate, glucose as well as the desired reactionproduct rhamnose remain in the filtrate.

The isolation of the rhamnose remaining in the filtrate can be achievedby means of known processes, for example by ultrafiltration, by passingthe filtrate over cation and/or anion exchangers, by crystallization andby means of mechanical separation, such as filtration. Glucose which maybe present in the filtrate can also be removed, for example, by yeastfermentation.

The substances obtained in the work-up steps, such as the organicsolvents, the enzyme or the buffer, for example sodium citrates, can berecirculated and thus used for further reactions.

The analysis of the reaction products can be carried out by HPLC, e.g.using standard HPLC equipment and columns containing reverse-phasematerials with a C₁₈-alkyl coating.

The following examples are intended to illustrate the present invention.However, they are in no case to be considered as limiting.

EXAMPLES

The supply sources for the substances used are as follows:

Rutin: Merck KGaA, Item No. 500017

Naringinase: Sigma, Item No. N-1385

Hesperidinase Amano, Item No. HPV 12519

Citric acid monohydrate Merck KGaA, Item No. 100243 Sodium hydroxideMerck KGaA, Item No. 105587 solution. Methyl acetate Merck KGaA, ItemNo. 809711

The reaction is checked by means of thin-layer chromatography (TLC) andthe reaction products are analyzed by means of HPLC.

TLC Conditions

Precoated TLC plates: Silica gel 60 (Merck KgaA, Item No. 105719),

Eluent: mixture of ethyl acetate ethyl methyl ketone: formic acid:water: 1-butanol in the volume ratio 50:30:10:10:5,

Spray reagent: iodine/sulfuric acid,

Detection: UV light (254 nm),

R_(f) values:

rutin: 0.38,

isoquercetin: 0.61,

quercetin: 0.96.

HPLC Conditions using a Standard HPLC Unit

Cartridge: LiChroCart® 2504/4 with

Column: LiChroSorb® RP18 (reversed phase material with C₁₈-alkyl coatingand a particle size of 5 μm (Merck KGaA, Item No. 151355)),

Eluent: mixture of acetonitrile and water in the volume ratio 20:80 (pH2; buffered with NaH₂PO₄•H₂O/H₃PO₄),

Flow: 1 ml/min,

Wavelength 260 nm,

Temperature: 30° C.,

Sample volume: 10 μl,

Sample preparation: dissolve 5 mg of the sample in 3 ml of methanol andmake up to 10 ml with the eluent,

Reaction times:

rutin: 7-7.5 min,

isoquercetin: 8.5-9 min,

quercetin: 40-43 min.

Example 1

3.15 g of citric acid monohydrate are dissolved in 150 ml of completelydeionized water and adjusted to a pH of 6.6 using 10 g of 32% aqueoussodium hydroxide solution 150 ml of methyl acetate are subsequentlyadded and 5.0 g of rutin and 0.5 g of naringinase are introduced under anitrogen atmosphere with stirring (200 revolutions/minute). The reactionmixture is then stirred at a reaction temperature of 40° C. for 24 h.After customary work-up, rhamnose and 3.82 g of yellow crystals areobtained. The analysis of the yellow crystals by means of HPLC resultsin the following composition:

Rutin: 1.2 area percent,

Isoquercetin: 94.4 area percent,

Quercetin: 2.6 area percent.

Example 2

0.32 g of citric acid monohydrate is dissolved in 150 ml of completelydeionized water, and 150 ml of methyl acetate are added. The emulsion issubsequently adjusted to a pH of 5.0 using 2.5 g of 1 normal aqueoussodium hydroxide solution, and 5.0 g of rutin and 0.125 g ofhesperidinase are introduced under a nitrogen atmosphere. The reactionmixture is then stirred (250 revolutions/minute) at a reactiontemperature of 40° C. for 21 h. After customary work-up, rhamnose and3.41 g of yellow crystals are obtained. The analysis of the yellowcrystals by means of HPLC results in the following composition:

Rutin: 0.1 area percent,

Isoquercetin: 98.0 area percent,

Quercetin: 0.2 area percent.

Example 3

6.37 g of citric acid monohydrate are dissolved in 300 ml of completelydeionized water and adjusted to a pH of 6.6 with 11.33 g of 32% aqueoussodium hydroxide solution. 300 ml of methyl acetate are subsequentlyadded and 20.11 g of a starting material mixture which consists of 53.5area percent of rutin, 39.8 area percent of isoquercetin and 0.4 areapercent of quercetin (mother liquor residue from rutin production), and1.11 g of naringinase are introduced under a nitrogen atmosphere. Thereaction mixture is then stirred (200 revolutions/minute) at a reactiontemperature of 40° C. for 46 h. After customary work-up, rhamnose and14.18 g of yellow crystals are obtained. The analysis of the yellowcrystals by means of HPLC results in the following composition:

Rutin: 0.5 area percent,

Isoquercetin: 92.0 area percent,

Quercetin: 4.7 area percent.

Comparison Example

12.6 g of citric acid monohydrate are dissolved in 600 ml of completelydeionized water and adjusted to a pH of 6.6 with 40 g of 32% aqueoussodium hydroxide solution. 10.0 g of rutin and 1.0 g of naringinase aresubsequently introduced under a nitrogen atmosphere with stirring (200revolutions/minute). After stirring at 36° C. for about 24 hours,isoquercetin and rutin are present in the reaction mixture in a ratio ofabout 2:1. The reaction mixture is stirred at 36° C. for a further 7 hand at 40° C. for 22 h and then cooled to 15° C. After customarywork-up, rhamnose and 7.25 g of yellow crystals are obtained. Theanalysis of the yellow crystals by means of HPLC results in thefollowing composition:

Rutin: 12.1 area percent,

Isoquercetin: 76.6 area percent,

Quercetin: 10.5 area percent.

The Comparison Example shows that on use of water alone as solvent lesssolid (yellow crystals) is obtained, which moreover contains morestarting material and more by-products than on use of a solvent mixturewhich consists of water and an organic solvent.

What is claimed is:
 1. A process for the enzymatic cleavage ofrutinosides to obtain rhamnose and/or the correspondingglucopyranosides, characterized in that the reaction is carried out inthe presence of a solvent mixture of water and one or more organicsolvents.
 2. The process as claimed in claim 1, characterized in thatthe reaction is carried out at a reaction temperature of 15 to 80° C. 3.The process as claimed in claim 1, characterized in that the reaction iscarried out at a pH from 3 to
 8. 4. The process as claimed in claim 1,characterized in that the pH is adjusted with the aid of a buffersystem.
 5. The process as claimed in claim 4, characterized in that thepH is adjusted. with the aid of aqueous citrate buffer.
 6. The processas claimed in claim 1, characterized in that the reaction is carried outin the presence of one or more of the organic solvents acetic acidesters, methanol, ethanol, methyl tert-butyl ester, toluene.
 7. Theprocess as claimed in claim 6, characterized in that the reaction iscarried out in the presence of one or more acetic acid esters.
 8. Theprocess as claimed in claim 7, characterized in that the reaction iscarried out in the